The role of neutral metalloproteinases in the maintenance of normal body functions, such as bone remodeling and wound healing, requires tight regulation. This regulation is altered in malignancy where metalloproteinases are necessary for tumor neoangiogenesis and invasion.
One example of ectopic activity of an otherwise normal function is collagenolytic degradation of basement membrane and extracellular matrix by secreted matrix metalloproteinases (MMPs).
Proteolysis and interruption of the basement membrane requires activation of specialized matrix metalloproteinases which selectively degrade basement membrane collagens type IV and V, the type IV collagenases or gelatinases (MMPs). Liotta, et al., Nature 284:67-68 (1980). Two species of MMPs, the 72 kDa species (MMP-2, gelatinase A) and the 92 kDa species (MMP-9, gelatinase B) have been isolated, cloned and sequenced. Liotta, et al., ibid.; and Liotta, et al., Biochemistry 20:100-104 (1981). Both MMPs are secreted as latent proenzymes which require the removal of an 80 or 87 amino acid amino-terminal domain for activation. Stefler-Stevenson, et al., J. Biol Chem. 264:1353-1356 (1989). Little is known about the signaling pathways which mediate the production and activation of these enzymes. While the activity of these proteinases is metal ion (Zn.sup.++)-dependent, the regulation of MMP production by divalent cations is unknown.
Recent studies have focused on the induction and activation of MMPs. Others have demonstrated that stable transfection of primary rat embryo fibroblast cells with activated Ha-ras resulted in the metastatic phenotype and increased production of type IV collagenases; this result was abrogated by simultaneous transfection of adenovirus E1A with Ha-ras (Pozzatti, et al., Science. 23:223-227 (1986)). Similar results were seen when Ha-ras was transfected into human bronchial epithelial cells (Collier, et al., J. Biol. Chem. 263:6579-6587 (1988)). Further investigation into the induction and subsequent regulation of MMP-2 demonstrated coordinate regulation of MMP-2 expression and function by treatment of human melanoma and fibrosarcoma cells with transforming factor-.beta..sub.1 (TGG-.beta..sub.1), 12-O-tetradecanoylphorbol-13-acetate (TPA), interleukin-1 (IL-1), and retionic acid (RA). See, Brown, et al., Cancer Research 50:6184-6191 (1990). TGF-.beta..sub.1 treatment resulted in induction of MMP-2 and inhibition of the interstitial collagenase, MMP-1. Conversely, TPA treatment produced inhibition of MMP-2 with induction of MMP-1. These results suggested involvement of transcriptional regulation and protein kinase C second messenger signaling pathways in the production of MMP-2. To date, there has been no demonstration of calciumdependent regulation of MMP-2 production or activation.
The action of the endogenous activator of protein kinase C, diacylglycerol, is mimicked by phorbol esters such as TPA (Castagna, et al., J. Biol. Chem. 257:7847 (1982)). Diacylglycerol is produced by hydrolysis of membrane phospholipids, such as phosphatidylinositol hisphosphate. Phosphatidylinositol hisphosphate is hydrolyzed to diacylglycerol and inositol trisphosphate by phospholipase C (PLC) -.beta. and -.gamma.. These enzymes are regulated through different signal transduction pathways. PLC-.beta. is stimulated in response to ligand binding to seven transmembrane domain receptors which associate with guanine nucleotide binding protein intermediates (Berridge, et al., Nature 341:197-205 (1989)). In most instances, this activation is calcium-independent. Receptor tyrosine kinase stimulation by ligand binding may cause activation of PLC-.gamma. by specific tyrosine phosphorylation (Aaronson, Science 254:1146-1153 (1991)). This process may be dependent upon either calcium influx or intracellular calcium mobilization (Gusovsky, et al., J. Biol. Chem. 268:7768-7772 (1993); Tanaguchi, et al., J. Biol. Chem. 268:2277-2279 (1993); and Chapron, et al., Biochem. Biophs. Res. Comm. 158:527-533 (1989)). The activated phospholipases C then hydrolyze membrane phosphatidyl inositol bisphosphate to yield diacylglycerol and inositol polyphosphates, both which may act as second messengers. Diacylglycerol may be involved in vivo with activation of matrix metalloproteinases as suggested by the phorbol ester experiments or it may be the upstream activator of other signaling molecules such as mobilization of calcium.
Compound 1, shown below, is a novel calcium influx inhibitor with antiproliferative and antimetastatic activities (Kohn, et al., J. Natl. Cancer Inst. 82:54-60 (1990); Felder, et al., J. Pharm. Exp. Therapeut. 257:967-971 (1991); and Kohn, et al., Cancer Res. 52:3208-3212 (1992)). Investigation into the mechanism of action of compound 1 has demonstrated that it inhibits receptor-operated and voltage-gated calcium influx (Felder, et al., J. Pharm. Exp. Therapeut. 257:967-971 (1991); Hupe, et al., J. Biol. Chem. 266:10136-10142 (1991)), calcium-dependent arachidonic acid release (Felder, et al., J. Pharm. Exp. Therapeut. 257:967-971 (1991); Clark, et al., Cell. 65:1043-1051 (1991)) and tyrosine phosphorylation with activation of phospholipase C-.gamma. (Gusovsky, et al., J. Biol. Chem. 268:7768-7772 (1993)). These functions could also be inhibited by compound 2, shown below, a chemically different inhibitor of receptor operated calcium channels. See, Gusovsky, et al., J. Biol. Chem. 268:7768-7772 (1993); Merritt, et al., J. Biol. Chem 271:515-522 (1990). This confirmed the role of compound 1-mediated inhibition of calcium influx on these biochemical events. The ability of compound 1 to inhibit selected calcium-mediated signal transduction pathways made it an ideal tool with which to investigate the role of calcium regulation underlying the expression and activation of MMP-2. ##STR1##